Methods of Treating and Preventing Neurological Symptons Caused by Avian Reovirus and Novel Associated Characteristics

ABSTRACT

Embodiments of the present invention generally relate to novel methods for the treatment and/or prevention of neurological symptoms caused by an avian reovirus, enteric reovirus strain (ERS), and associated characteristics. Other embodiments generally comprise and immunogenic composition or vaccine comprising an ERS for the treatment and/or prevention of neurological symptoms.

RELATED APPLICATIONS

This application claims priority from provisional application No.60/417,245, filed on Oct. 9, 2002, provisional application No.60/418,589, filed on Oct. 15, 2002, provisional application No.60/424,163, filed on Nov. 6, 2002, and provisional application No.60/435,192, filed on Dec. 20, 2002.

FIELD OF THE INVENTION

The present invention is related to methods of treating and/orpreventing neurological symptoms caused by avian reovirus and novelassociated characteristics.

BACKGROUND OF THE INVENTION

Reovirus infections are prevalent worldwide in poultry and have beenisolated from chickens showing a wide variety of clinical signsincluding viral arthritis/tenosynovitis, malabsorption syndrome (MAS),pericarditis, myocarditis, and immunosuppression [McNulty et al., “Virusinfection of birds,” Reovirus, Chapter 13, pp 181-193 (1993)].

Viral arthritis/tenosynovitis particularly in broiler breeds is the mostimportant disease attributable to reovirus infection. [Nibert et al.,Fields Virology 4: p. 1681 (2001)]. It is a persistent viral infectionwith chronic inflammatory lesions, confined mainly to the hock jointsand leg tendons of the chicken. The lesions are localized to synovialstructures [Menendez et al., Avian Dis. 19: 112-117 (1975); Ellis et al,Avian Dis. 27: 644-651 (1983); Kibenge et al, Avian Pathol. 14: 87-98(1985)]; [Nibert et al., Fields Virology 4: p. 1681-1682 (2001)].

Clinical signs/symptoms of reovirus infection have also been associatedwith malabsorption syndrome (MAS) by several groups [Kouwenhoven et al.,Avian Pathol. 7: 183-187 (1978); Page et al., Avian Dis. 26:618-624(1982); Hieronymus et al., Avian Dis. 27: 255-260 (1983); Goodwin et al,Avian Dis. 37: 451-458 (1993)]. The syndrome is characterized by weightgain depression with non-uniform growth, defective feathering anddiarrhea with undigested food and watery content. The pathologic changesmostly include proventriculitis enteritis, pancreatitis and hepatitis.MAS may be caused either by maldigestion or malabsorption or both.Lesions that are found can cause an impairment of digestion byinsufficiency of digestive secretions and or an impairment of absorptionbecause of insufficient absorptive capacity. However, there are avariety of pathogenicities of reovirus isolated from MAS as can beenseen in the variations of depressed weight gains. [Nibert et al., FieldsVirology 4: p. 1681-1682 (2001); Page et al., Avian Dis. 26:618-624(1982); McNulty et al., Avian Pathol. 13: 429-439 (1984); Decaessteckeret al., Avian Pathol. 15: 769-782 (1986); Kibenge and Dhillon, AvianDis. 31: 39-42 (1987); Kouwenhoven et al., Avian Pathol. 17: 879-892(1988); Montgomery et al., Avian Dis. 41: 80-92 (1997) Songserm et al.,Avian Dis. 46: 87-94 (2002)]. To date, there has been no disclosure inthe prior art of an avian reovirus causing neurological symptoms.

There are several prior art vaccines for known avian reoviruses.Examples of live vaccines include strain S1133 and 2177. Live vaccinesin the United States have been developed from various passage levels ofavian reovirus strain S1133, isolated and characterized by van der Heidefrom a field case of tenosynovitis. Strain S1133 was grown serially 235times in the chorioallantoic membrane (CAM) at 37.degree. C. and then 65times in chicken embryo fibroblast (CEF) at 32.degree. C. An additional135 passages were carried out at 37° C. in CEF [van der Heide et al.,Avian Dis., 27:698-706 (1983)]. Strain 2177 was isolated andcharacterized by Rosenberger from a field case of avian reovirus [U.S.Pat. No. 5,525,342]. Strain 2177 was isolated from chickens exhibitingreovirus disease by inoculating tissue samples into embryonated eggs andcollecting the yolk fluid. This yolk fluid was then inoculated into CEFand passed 14 times until the cytopathology was observed at which timethe virus was plaque purified. This yolk fluid was then propagated inembryonated eggs to produce a stock of virus. The stock was found to benon-pathogenic and was titled strain 2177. To date, no reovirus has beenable to be grown/cultured immediately upon Vero cells withoutadaptation. [Nibert et al., Fields Virology 4: pp. 1681-1682 (2001)].The most common method is propagation in embryonated chicken eggs. Thereis disclosure in the prior art of an orthoreovirus from an Australianflying fox, a mammalian reovirus, having some characteristics of anavian orthreovirus (reovirus) that is able to be propagated in mammaliancells. [Nibert et al., Fields Virology 4: p. 1682 (2001)]. However,there is no disclosure in the prior art of an avian reovirus that isable to be propogated in a mammalian cell without adaptation. [Nibert etal., Fields Virology 4: p. 1682 (2001)]. As well, the prior art does notdisclose a method of treating and/or preventing neurological symptoms inpoultry caused by an avian reovirus. Accordingly, the art field is insearch of a method to treat and/or prevent neurological symptoms causeby an avian reovirus.

Recently, van Loon et al [Veterinary Quarterly, 23: 129-133 (2001)]described the isolation and identification of a new class of reoviruses,the so-called enteric reovirus strains (ERS). ERS was isolated frombroilers in showing high mortality. Those broilers came from parentflocks that were well vaccinated against reovirus infections. It becameclear that ERS was a new serotype of reovirus since the virus could notbe neutralized by known reoviruses using the plaque reduction test [vanLoon et al., Veterinary Quarterly, 23: 129-133 (2001)]. Furthermore,characterization of ERS with a panel of monoclonal antibodies revealedthat these strains showed a distinct panel pattern compared to reovirusstrains that have been described in the literature previously[Hieronymus et al., Avian Dis. 27: 246-254 (1983); Johnson, Avian Dis.16: 1067-1072 (1972); Olson et al., Am J Vet Res. 18: 735-739 (1957);Rosenberger et al, Avian Dis. 33: 535-544 (1989); van der Heide et al.,Avian Dis. 18: 289-296 (1974)]. The novel antigenic class of reoviruswas further identified by the art-accepted practice of a plaquereduction assay, U.S. patent application No. An explanation of a plaquereduction assay may be found in an article by Nersessian et al. (J. Vet.Res. N50, 1989, pp. 1475-1480). The article confirms the heterogenicimmunological character of avian reovirus and validates the use ofplaque reduction assays for determining and characterizing antigenicrelationships (including, but not limited to, similarities anddifferences) between reovirus isolates. Upon screening for Reoviruses inthe field, it was observed that ERS type strains were also present inThe Netherlands, Belgium, Ireland, United Kingdom, Spain, Germany,Italy, USA, Argentina, United Arabic Emirates, South Africa, ThePhilippines and Indonesia. These ERS type strains were usually isolatedfrom birds with MAS. Studies of the pathogenicity and dissemination ofERS in specific pathogen free (SPF) chickens demonstrated that ERS wasable to induce a high mortality, tenosynovitis (unpublishedobservations) and MAS [van Loon et al., Veterinary Quarterly, 23:129-133 (2001)]. Also in commercial broilers with maternally derivedantibodies against reovirus, van Loon et al [Veterinary Quarterly, 23:129-133 (2001)] showed a growth retardation of respectively 35% and 25%in broilers inoculated at day old or at 7 days old. Further study andevaluation of this novel class has yielded other surprisingcharacteristics.

SUMMARY OF THE INVENTION

Generally, embodiments of the present invention relate to a class ofavian reovirus that causes neurological symptoms and methods of treatingand/or preventing neurological symptoms caused by the class of avianreovirus.

In an embodiment, it has been discovered that the class of reovirusreferred to as avian Enteric Reovirus Strain (ERS) causes neurologicalsymptoms. Such neurological symptoms comprise, but are not necessarilylimited to, twisted neck, tremors, and/or the like. Further, it has beendiscovered, in an embodiment, that a method of treating and/orpreventing neurological symptoms caused by an avian reovirus comprisesadministering an effective amount of an immunogenic composition orvaccine comprising an ERS avian reovirus in a live, attenuated or killedform and a carrier or diluent.

In an embodiment of a method of the present invention, the immunogeniccomposition or vaccine administered comprises reoviruses of an antigenicclass of reovirsuses defined as having the characteristics ofembodiments of the class of virus of U.S. application Ser. No.09/493,484 (hereinafter referred to as the '484 application). The '484application claims priority from published European Patent applicationnumber 00200256.6, filed on Jan. 25, 2000. The European Patentapplication published on Aug. 2, 2000 under publication number EP 1 024189 A1.

Such class of reovirus is defined as belonging to the class of reovirusthat is able to induce antiserum in an animal, which antiserum causes areduction of the plaques formed by strain ERS, a sample of which isdeposited at the ECACC, Salisbury, UK, under accession no. 99011475, ofat least 75% in a plaque reduction assay, and, such class is furtherdefined by reactivity in an IFT with a polyclonal antiserum raisedagainst an avian reovirus isolate, preferably against the prototypereovirus strain 1133, and the absence of reactivity in the IFT with theMoabs INT 13-06, INT 14-11 and 15-01 INT (hybridomas of which aredeposited at the ECACC under accession no. 99011472, 99011473 and99011474, respectively).

Nothing in this summary should be construed as limiting the scope of theinvention. For a further understanding of the scope of invention,attention should be had to the following detailed description, examplesand claims.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “relatively non-pathogenic” means and refers toa significant reduction in the pathology of a virus, such that less thanor equal to about 20% of poultry immunized with a live strain of thevirus would be affected by the virus. As used herein, the term “chicken”means and refers to all chickens, including broilers, reproductionstock, laying stock and the like. The term “poultry,” includes, but isnot limited to, chickens, turkeys, water fowl, guineas, quail, pigeons,ostriches, and the like. As used herein, the term “naturally” meanswithout human intervention. However, it is contemplated that a“naturally non-pathogenic” strain may be passaged in various embodimentsto prepare a virus stock and the like. It is further contemplated thatsuch passaging does not cause a virus to be considered other than“naturally non-pathogenic” if the virus was “relatively non-pathogenic,”as defined, before passaging.

As used herein, the term “vaccine strain” means and refers to a viralstrain suitable for use in an immunogenic composition or vaccine. A“vaccine strain” can comprise, but is not necessarily limited to, anon-pathogenic strain or relatively non-pathogenic strain, a killedstrain, and/or an attenuated strain.

The '484 application, filed on Jan. 28, 2000 defined, for the firsttime, a novel antigenic class of avian reovirus. The '484 applicationclaims priority from published European Patent application number00200256.6, filed on Jan. 25, 2000. The European Patent applicationpublished on Aug. 2, 2000 under publication number EP 1 024 189 A1.

The novel class in the application was characterized as avian reovirusERS (enteric reovirus strain). The class was identified as ERS becausethe class was found to cause enteric disorders, a reovirus associateddisease, such as too liquid feces and/or maldigested food. Now,surprisingly, it has been found that the class of reovirus, the avianreovirus ERS, causes neurological symptoms. The neurological symptomsinclude, but are not limited to, nor require all, twisted neck (alsoreferred to as torticollosis) and tremors.

Accordingly, embodiments of the present invention generally relate to aclass of avian reovirus that causes neurological symptoms and methodsfor preventing neurological symptoms caused by an avian reovirus. In anembodiment, methods of the present invention comprise administering aneffective amount of an immunogenic composition or vaccine comprising anavian reovirus that causes neurological symptoms in a live, attenuatedor killed form and a carrier or diluent. Therefore, further embodimentsare directed towards an immunogenic composition or vaccine comprisingreovirus of an antigenic class of reovirsuses having the characteristicsof the class of virus of European Application EP 1 024 189 A1 .

Such class of reovirus is defined as belonging to the class of reovirusthat is able to induce antiserum in an animal, which antiserum causes areduction of the plaques (plaque reduction assay) formed by strain ERS,a sample of which is deposited at the ECACC, Salisbury, UK, underaccession no. 99011475, of at least 75% in a plaque reduction assay,and/or, such class is further defined by reactivity in an IFT with apolyclonal antiserum raised against an avian reovirus isolate,preferably against the prototype reovirus strain 1133, and the absenceof reactivity in the IFT with the Moabs INT 13-06, INT 14-11 and 15-01INT (hybridomas of which are deposited at the ECACC under accession no.99011472, 99011473 and 99011474, respectively).

Other reovirsues of the present invention and for use invaccines/methods of the present invention are characterized in that thereovirus of the immunogenic composition or vaccine that belongs to theclass of reovirus that is able to induce antiserum in an animal, whichantiserum causes a reduction of the plaques formed by strain ERS of atleast 80% in a plaque reduction assay. Other reoviruses of the presentinvention and for use in vaccines/methods of the present invention arecharacterized in that the reovirus of the immunogenic composition orvaccine that belongs to the class of reovirus that is able to induceantiserum in an animal, which antiserum causes a reduction of theplaques formed by strain ERS of at least 90% in a plaque reductionassay.

Exemplary, non-limiting, strains representative of reoviruses suitablefor use in methods of the present invention include, but are not limitedto, strain ERS (isolate 1), deposited at the ECACC, Salisbury, UK, underaccession no. 99011475; strain ERS 1037, deposited at the ATCC,Manassas, Va. 20108, U.S.A. on Oct. 1, 2002, under accession no.pta-4735; strain ERS 060E, deposited at the ATCC, Manassas, Va. 20108,U.S.A. on Oct. 30, 2002, under accession no. pta-4782; and, strain ERS074, deposited at the ATCC, Manassas, Va. 20108, U.S.A. on Oct. 30,2002, under accession no. PTA-4783.

Strains of the present invention and strains used in immunogeniccompositions or vaccines for methods of the present invention may beisolated from poultry as is common in the art. For chickens, a strain ofreovirus is isolated from chickens with reovirus associated disease.Processes for virus isolation are common in the art. In an isolation,the virus was isolated from infected tendons collected from broilersshowing signs of reovirus associated disease. However, methods of thepresent invention are not limited by from what part of avian the virusis isolated.

In fact, surprisingly, it has been found that avian reovirus of thepresent invention may be isolated from the neurological system ofpoultry suffering from avian reovirus induced neurological symptoms,such as from the brain and the spinal chord. Avian reovirus has notpreviously been isolated from the brain, spinal chord, and/or otherstructures associated with the neurological system. Further, it isbelieved that only strains causing neurological symptoms may be isolatedfrom the brain, spinal chord, and/or other structures associated withthe neurological system. In fact, it is illustrated in the Experimentalsection that prior art reoviruses not causing neurological symptomscannot be isolated from the brain, spinal chord, and/or other structuresassociated with the neurological system.

Accordingly, the present invention discloses a class of avian reovirusesthat may be isolated from the neurological system of poultry. In variousembodiments, the class of avian reovirus so characterized may beisolated from the brain, spinal chord, and/or other structuresassociated with the neurological system.

In various embodiments, immunogenic compositions or vaccines used withmethods of the present invention may be administered alone or incombination with other viral vaccines for poultry, such as those forMarek's Disease Virus, Infectious Bursal Disease Virus, NewcastleDisease Virus, Infectious Bronchitis Virus, Avian EncephalomyelitisVirus, Fowl Pox Virus, Chicken Anemia Agent and/or the like. Otherembodiments may combine bacterial antigens and the like. Further,preferred immunogenic compositions or vaccines used with methods of thepresent invention are vaccines which may be administered at a young age(e.g., one day) and/or in ovo.

Administration of immunogenic compositions or vaccines in methods of thepresent invention may be by any route commonly used for avian reovirusadministration Preferred routes of administration include massapplication routes, such as drinking water (orally) and spray. However,other embodiments of administration may include injection and the like.

It is common in administration by the drinking water route to deprivethe animals of water for about 2 to 4 hours before placing theimmunogenic composition or vaccine containing water before them. It isimportant that there is enough drinker space for all birds to drinkevenly to allow for even vaccine dispersion. The vaccine is applied infresh drinking water at an effective amount, a concentration calculatedto give each bird a sufficient dose for an immunogenic composition orvaccine.

In various embodiments, in order to prevent a reduction of the viablevaccine virus by the presence of small amounts of chlorine, iron, zincor copper ions in the drinking water and/or to prevent inactivation ofthe live vaccine virus because of increased concentration of dissolvedsalts as a result of desiccation of the drinking water, small amounts ofa protein protectant, such as skimmed milk, skimmed milk powder orgelatin can be added to aqueous phase.

In various embodiments utilizing a spray method, common methods ofapplication include, but are not limited to, a coarse spray applicationand an aerosol administration. In the coarse spray method particlesusually have an initial droplet size ranging from 10 to 100 microns andin the aerosol administration method droplets usually range from <1 to50 microns. However, any droplet size may be used in varying embodimentsof the invention.

It is common in the industry to utilize conventional spray-apparatus andaerosol generators for the generation of the small particles, such asthe commercially available spray generators for knapsack spray, hatcheryspray and atomist spray. Details concerning conventionalspray/aerosol-and drinking water vaccination can be found in the“Compendium, administration of poultry vaccines” issued by theGezondheidsdienst voor Pluimvee, Doom, The Netherlands, van Eck et al.,VI-VII, 1988.

Further embodiments of a method of the present invention may utilizeadministration of a vaccine through eye drop and/or beak dipping and/orany other method(s) common in the art.

Other embodiments of the present invention may utilize administration ofan inactivated avian reovirus. Inactivated forms potentially provide abenefit of elevated levels of protective antibodies for a long duration.This property makes an inactivated vaccine particularly well suited forbreeder vaccination, in certain instances.

As is known in the art, an aim in the inactivation of viruses harvestedafter propagation is to eliminate reproduction of viruses. In general,this can be achieved by chemical or physical means. Chemicalinactivation can be effected by treating the viruses with, for example,enzymes, formaldehyde, β-propiolactone, ethylene-imine, or a derivativethereof. If necessary, the inactivating material is neutralizedafterwards. For example, material inactivated with formaldehyde can beneutralized with thiosulphate. Physical inactivation can preferably becarried out by subjecting the viruses to energy-rich radiation, such asUV light or γ-rays. If desired, after treatment the pH can be adjustedto a value of about 7.

Inactivated vaccines may be administered parent rally, e.g.intramuscularly, subcutaneously, or any other method common in the art.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and the appended Claims are intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth whether now existing or afterarising. Further, while embodiments of the invention have been describedwith specific dimensional characteristics and/or measurements, it willbe understood that the embodiments are capable of different dimensionalcharacteristics and/or measurements without departing from theprinciples of the invention and the appended Claims are intended tocover such differences. Furthermore, all patents, patent applications,articles, and other publications mentioned herein are herby incorporatedby reference.

For a further understanding of various embodiments of the presentinvention, reference should be had to the following examples:

EXAMPLES

Samples from flocks of chickens in the United States of America weretaken and isolated into strains. Certain of these strains wereidentified as ERS 060E, ERS 074, and ERS 1037. The flocks from whichthese chickens were taken were experiencing classic symptoms of ERS,including, but not limited to enteritis. Serum from certain chickens wassent to Boxmeer, The Netherlands for a panel pattern to confirm that thechickens belonged to the antigenic class identified in EP 1 024 189 A1.All three sera illustrated the absence of reactivity in an IFT withMoabs INT 13-06, INT 14-11, and 15-01 INT.

Based upon the results comprising the study, it was determined thatstrains ERS 1037, ERS 060E, and ERS 074 belonged to the novel antigenicclass of reoviruses identified as ERS in EP 1 024 189 A1.

Further studies of strains ERS, ERS 060E and ERS 074 revealed that thenovel antigenic class of reoviruses causes neurological symptoms ininfected poultry. Such neurological symptoms including, but not limitedto twisted neck, tremors and/or the like. The observations were made inboth SPF chickens and commercial broilers, with maternally derivedantibodies against reovirus, after infection with the novel antigenicclass of reovirus, ERS.

In one example, when chickens were inoculated via the foot pad routeand/or subcutaneous at one day old, the survivors developed nervoussymptoms such as tremors and twisted neck. These symptoms can be seenafter inoculation.

In the field, ERS was isolated from chickens showing neurologicalsymptoms. These neurological symptoms could be reproduced underexperimental conditions with the isolated ERS strain.

Example 1

Ongoing studies have revealed that strains of avian reovirus causingneurological symptoms can be isolated from the brain and/or spinalchord.

In one example, ERS 074 was reisolated from SPF Chickens' brains andspinal chords using the following procedure:

1. Chickens showing neurological symptoms were necropsied and the headand necks were collected and frozen. A chicken was determined to beshowing neurological symptoms when the chicken was observed with twistedneck and/or tremors.

2. Heads and necks were thawed.

3. Brain and upper portion of the spinal cord were asceptically swabbed.

4. Swabs were placed in TPB and vortexed.

5. Samples were frozen/thawed three times.

6. Samples ere centrifuged for 10 minutes at approx. 3000 rpm.

7. Supernatant was removed and filtered through 0.45 μm and 0.2 μmsyringe filters.

8. 0.1 ml of filtered material was inoculated into 10 day eggs using thedrop CAM route.

9. Eggs were candled daily for death.

10. Six days post inoculation all eggs were opened.

11. Liver and chorioallantoic membranes were harvested from infectedembryos.

12. Liver and chorioallantoic membranes were homogenized using a Waringblender.

13. Homogenates were frozen and thawed three times.

14. An AGP(Agar Gel Precipitin Test) was used to detect avian reovirusantigen.

The AGP test was positive for avian reovirus antigen.

Example 2 (Panel Pattern)

Polyclonal antiserum was prepared by infecting rabbits (1-1.5 kg) withpurified avian reovirus strain 1133. Booster injections took place 28and 84 days after the first injection. Blood was collected and serumisolated 14 days after the last injection.

Different reovirus were characterized with different Moabs. In anembodiment, primary CEL cells were grown in 96-well polystyrenemicrotitre plates. Uninfected cells served as controls. After 2-4.daysof incubation at 37° C. with 5% CO₂, infected monolayers were fixed withcold 96% ethanol. The alcohol was discarded and the plates were washedwith washing buffer and 100 μl of different hybridoma cell culturesupernatant diluted 1:50 or 1:200 in PBS or 100 μl of rabbit polyclonalserum (rabbit 68A) diluted 1:50, was added to each well. The plates wereincubated for 60-90 minutes at 37° C., washed twice with washing bufferand reacted with 1:100 diluted fluorescent isothiocyanate-labelledrabbit anti-mouse or 1:100 diluted isothiocyanate-labelled goatanti-rabbit serum. The plates were then washed and fixed with aglycerol/PBS solution (1:1). The presence of fluorescence was observedwith a fluorescence microscope.

The antiserum panel used in this experiment consisted of the followingpolyclonal antiserum and Moabs raised against the prototype avianreovirus strain 1133:

Rabbit 68A rabbit polyclonal antiserum Moab 154 Vakharia et al. 1996(supra) Moab 14-67 Intervet International B.V. Moab INT 13-06 ECACCaccession no. 99011472* Moab INT 14-11 ECACC accession no. 99011473*Moab 15-01 INT ECACC accession no. 99011474*The panel pattern was as follows:

Strain Rabbit 68A 154 14-67 14-11 13-06 15-01 S-1133240817332177ERS(isolate 1)ERS 1037ERS 060EERS 074ERS 015

*hybridomas of the Moabs arte deposited

As can be seen from the panel patterns, strains ERS 1037, ERS 060E, andERS 074 have a comparable pattern to strain ERS (isolate 1) while beingdifferentiated from strains S-1133, 2408, 1733, and 2177.

Example 3

The following experiments demonstrate studies conducted with variousidentified strains.

Experiment 3a Experimental Design

Twenty SPF chickens were inoculated orally at day old with 0.5 ml plaquepurified strain ERS (isolate 1) (6.09 log₁₀TCID₅₀/bird). The chickenswere observed daily for clinical sign during 14 days. At 14 days of ageall chickens were slaughtered.

Results

35% of the chickens died during the first 6 days (Table 1). At day 10,one chicken developed neurological symptoms and at day 13 a secondchicken showed neurological symptoms. Further, chicks infected with ERSdemonstrated a 10.1 gram/day weight gain, while the control groupexperienced a 17.0 gram/day weight gain.

TABLE 1 Percentage mortality Days after inoculation Mortality (numberbirds) % Mortality 3 1 5 4 2 15 5 3 30 6 1 35

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds/remaining birds)* % NS 10 1/13 7.7 131/13 15 *A chicken was determined to be showing neurological symptomswhen the chicken was observed with twisted neck and/or tremors.

Experiment 3b

10 SPF chickens were inoculated subcutaneously with 0.2 ml at one day ofage with strain ERS 060E (10³ TCID₅₀/bird): The chickens were observedfor clinical sign at 15 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 20 2 20

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 20 4 40 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results.

20% of the birds died during the first 15 days. 40% of the birdssuffered from neurological symptoms. Further, chicks infected with ERS060E demonstrated an average 93.82 gram/day weight gain.

Experiment 3c

10 SPF chickens were inoculated subcutaneously with 0.2 ml at one day ofage with strain ERS 074 (10³ TCID₅₀/bird). The chickens were observedfor clinical sign at 20 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 20 3 30

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 20 0 0 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

30% of the birds died during the first 20 days. 0.0% of the birdssuffered from neurological symptoms. Further, chicks infected with ERS074 demonstrated a 126.38 gram/day weight gain.

Experiment 3d

10 SPF chickens were inoculated via the foot pad route with 0.2 ml atone day of age with strain ERS 060E (10³ TCID₅₀/bird). The chickens wereobserved for clinical sign at 20 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 20 2 20

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds) % NS 20 1 10 * A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

20% of the birds died during the first 20 days. 10% of the birdssuffered from neurological symptoms. Further, chicks infected with 060Edemonstrated a 87.81 gram/day weight gain.

Experiment 3e

10 SPF chickens were inoculated via the foot pad route with 0.2 ml atone day of age with strain ERS 074 (10³ TCID₅₀/bird). The chickens wereobserved for clinical sign at 20 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 20 10 100

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 20 5 50 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

100% of the birds died during the first 20 days. 50% of the birdssuffered from neurological symptoms. Further, chicks infected with ERS074 demonstrated a 94.37 gram/day weight gain.

Experiment 3f

20 broiler chickens were inoculated subcutaneously with 0.2 ml at oneday of age with strain ERS 060E (10³ TCID₅₀/bird). The chickens wereobserved for clinical sign at 14 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 14 3 15

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 14 2 10 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

15% of the birds died during the first 14 days. 10% of the birdssuffered from neurological symptoms. Further, chicks infected with ERS060E demonstrated an average weight of 543 grams at 21 dayspost-inoculation. Negative control birds had an average weight of 751grams at 21 days post-inoculation.

Experiment 3g

20 broiler chickens were inoculated subcutaneously with 0.2 ml at oneday of age with strain ERS 074 (10³ TCID₅₀/bird). The chickens wereobserved for clinical sign at 14 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 14 5 25

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 14 5 25 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

25% of the birds died during the first 14 days. 25% of the birdssuffered from neurological symptoms. Further, chicks infected with ERS074 demonstrated an average weight of 488 grams at 21 dayspost-inoculation. Negative control birds had an average weight of 751grams at 21 days post-inoculation.

Experiment 3h

20 broiler chickens were inoculated via the foot pad route with 0.2 mlat one day of age with strain ERS 060E (10² TCID₅₀/bird). The chickenswere observed for clinical sign at 14 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 14 5 25

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 14 1 5 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

25% of the birds died during the first 14 days. 5% of the birds sufferedfrom neurological symptoms. Further, chicks infected with ERS 060Edemonstrated an average weight of 442 grams at 21 days post-inoculation.Negative control birds had an average weight of 751 grams at 21 dayspost-inoculation.

Experiment 3i

20 broiler chickens were inoculated via the foot pad route with 0.2 mlat one day of age with strain ERS 074 (10² TCID₅₀/bird). The chickenswere observed for clinical sign at 14 days.

TABLE 1 Percentage mortality Days after inoculation Mortality (no. ofbirds) % Mortality 14 10 50

TABLE 2 Percentage neurological symptoms Neurological Symptoms Daysafter inoculation (number birds)* % NS 14 1 5 *A chicken was determinedto be showing neurological symptoms when the chicken was observed withtwisted neck and/or tremors.

Results:

50% of the birds died during the first 14 days. 5% of the birds sufferedfrom neurological symptoms. Further, chicks infected with ERS 074demonstrated an average weight of 473 grams at 21 days post-inoculation.Negative control birds had an average weight of 751 grams at 21 dayspost-inoculation.

Experiment 3j Experimental Design

Seventy-five SPF chickens and seventy-five commercial broilers withmaternal antibodies against reovirus (MDA⁺) were divided in 5 groups of15 birds. The birds received plaque purified ERS strain isolate 1 orallyor SC at 1 or 7 days of age. Table 1 shows the experimental design indetail. The chickens were observed daily for clinical sign during 7weeks. At 7 weeks of age all remaining chickens were slaughtered.

TABLE 1 Experimental design Age of Dose Immune inoculation Route of(log₁₀TCID₅₀/ Group status (days) inoculation bird) 1 SPF 1 Oral 5.2 2SPF 1 SC 4.9 3 MDA⁺ 1 Oral 5.2 4 MDA⁺ 1 SC 4.9 5 SPF 7 Oral 4.9 6 SPF 7SC 5.0 7 MDA⁺ 7 Oral 4.9 8 MDA⁺ 7 SC 5.0 9 SPF — — — 10  MDA⁺ — — —

TABLE 2 Percentage mortality Days after Mortality (number Groupinoculation birds) % Mortality 1 4-5 2 13.3 2 3-5 15 100 3 6-8 3 20 43-7 7 46.6 5 — 0 0 6 — 0 0 7 — 0 0 8 — 0 0 9 — 0 0 10 — 0 0

Results

Table 2 shows the percentage of birds that died after ERS inoculation.100% of the SPF birds and 46.6% of the MDA⁺ birds died after SCinoculation at 1 day of age within 3 to 7 days. The percentage mortalityafter oral inoculation at 1 day of age was 13.3 and 20% respectively forSPF and MDA⁺ chickens within 4 to 8 days. When birds were inoculated at7 days of age none of the birds died. Table 3 shows the percentagetwisted necks. In both SPF and MDA⁺ birds, neurological symptoms occurafter oral or SC inoculation at 1 or 7 days of age. The neurologicalsymptoms were seen from 6 days after inoculation.

TABLE 3 Percentage neurological symptoms Neurological Symptoms Daysafter (number birds/ Group inoculation remaining birds) % NS 1 — 0 0 2 —0 0 3 13 1/12 8.3 4 11 1/8  12.5 5 — 0 0 6  7 1/15 6.6 7 6-12 3/15 20 8— 0 0 9 — 0 0 10 — 0 0

Experiment 3k Experimental Design

Twenty-eight SPF chickens were inoculated SC at day old with 0.2mlplaque purified strain ERS Poland 2 (4.6 log₁₀TCID₅₀/birds). Thechickens were observed daily for clinical signs during 14 days. At 14days of age all chickens were slaughtered.

Results

96.4% of the chickens died during the first 6 days (Table 1). At day 9,one remaining chicken developed a twisted neck.

TABLE 1 Percentage mortality Days after infection Mortality (numberbirds) % Mortality 3 10 35.7 4 15 89.3 5 1 92.8 6 1 96.4

Experiment 31: Relatively Non-pathogenic Strains

ERS strain 1037 was passed in embryonated eggs for approximately 10passages to prepare a stock of virus that was used to inoculate 1-dayold broiler chickens via the intratracheal route. A study of ERS strain1037 was conducted against a highly pathogenic reovirus strain 1733.

Reo Strain 1037 Versus Reo Strain 1733

TABLE 1 Challenge results 14 day challenge results Total % ProgenyChall. # # positive # positive Posi- Affect- Group Strain chicks(weight)¹ (mortality)² tive ed SPF 1733 50 15 35 50 100 Controls 1037 5010 0 10 20 ¹Determined by t-test analysis ²Positive birds showedevidence of Reovirus infection at necropsy

Strain 1037 was shown to be relatively non-pathogenic because only about20% of the chickens were affected, exhibited any signs of malabsorptionsyndrome or growth depression, and none of the chickens died during thetest period. This is in contrast to 1733, also a malabsorption virus,that caused 100% of the chickens to be affected, to have weight lossand/or death. Likewise, as can be seen, strain 1733 killed 35% percentof the chicks whereas strain 1037 killed none. Therefore, strain 1037 isrelatively non-pathogenic, as herein defined.

Experiment 4: S1133 Infection of the Brains of Chickens after OralInoculation

Experimental Design:

10 SPF birds orally inoculated at day 1 with 2.6 log₁₀/bird S1133 virus

necropsy: 2 birds at 2, 3, 5, 7 and 10 dpc

isolation of reovirus from the brains using chickens embryo liver cells

Result:

No isolation of reovirus from the brains at any time.

Conclusion:

S1133, a prior art strain, does not infect the brains of chickens afteroral inoculation.

Experiment 5: Pathogenic Aspects of ERS Strains Virus Strain

ERS 015 was isolated from commercial broilers from well-vaccinatedparents in Poland in 1999. The birds suffered from high mortality and atnecropsy white spots on liver and hydropericarditis were found. Thisisolate was identified as being a Reovirus using IFT. Furthercharacterization with a panel of monoclonal antibodies confirmed thatthe isolate belonged to the ERS class. The isolate was plaque purifiedthree times and will be used as the challenge strain in the efficacystudies. Preliminary experiments showed that ERS 015 isolate was able toproduce tremor and twisted neck symptoms in 1-day old SPF birds. Apathogenesis study was conducted to confirm that the observed symptomswere caused by the new Reovirus strain.

Experimental Design

ERS 015 strain was plaque purified three times on CEF and used aschallenge strain. One hundred and thirty-two 1-day-old SPF birds weredivided in three groups. Group 1 had 55 birds and was inoculatedintramuscularly with 0.2 ml of ERS 015 (1.0 log₁₀ TCID₅₀/bird) at dayold. Group 2 had 55 birds and was inoculated orally with 0.2 ml of ERS015 (2.0 log₁₀ TCID₅₀/bird) at day old. Group 3 had 22 birds and wereinoculated intramuscularly with 0.2 ml PBS and served as negativecontrol birds.

All chickens were observed daily for the occurrence of clinical symptomsof disease. Special attention was taken for neurological symptoms, asherein defined.

At 7, 14 and 21 days after inoculation, blood was taken from birds thatwere sacrificed. Serum samples were examined for the presence ofantibodies against Reovirus strain ERS in the IFT, against Newcastledisease in the HI test, against infectious chickens encephalomyelitisvirus (AEV) in the ELISA test and against Marek's disease in the ELISA.

Two or three birds of groups 1 and 2 and one bird of group 3 weresacrificed at 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 and 21 days afterchallenge and examined for macroscopical lesions.

Samples of the brains were collected for virus isolation. The organswere placed in pycocrias tubes with glass beads and 1 ml of PBS+Penstrept (1000 μg U/ml). The mixtures were shaken for 20 minutes at thehighest speed in a Retch mixer mill. After homogenisation 0.1 ml of thesuspensions were inoculated on chicken embryo liver cells (CELi) (1*10⁶cells/ml in M6B8+0.1% NPPT+5% FCS). After 5 days incubation at 37° C.with 5% CO₂, all cultures were examined for specific REO CPE. If asuspension was negative a second inoculation on CEL was performed.

Samples of the brains, thoracic spinal cord, left and right sciaticnerves were collected for histological and immunohistochemicalexamination.

Immunohistochemistry was performed for detection of reovirus antigenusing monoclonal antibody 14-67 as primary antibody, a biotinylatedsecondary (rabbit-anti-mouse) antibody,streptavidin-biotine-enzyme-complex with alkaline phosphatase as enzyme,and new fuchsine as substrate.

Interpretation of the Results

Central nervous disorders were evaluated using the results of clinicalsymptoms, virus isolation and presence of viral antigen in the brains.If clinical symptoms were provoked by inoculation of ERS 015 and viruswas present from the brains, ERS was indicated as a causative agentinducing central nervous disorders such as tremor and twisted neck.

Results Clinical Symptoms

The results of the clinical signs of groups 1 and 2. No clinical signswere seen among birds of group 3. High mortality (80%) was seen within 7days after IM inoculation while only one birds died at 10 days afteroral inoculation. Poor growth and helicopter chickens were seen from day4 and chickens stayed small until the end of the experiment in bothgroups. In both groups, 1 bird developed tremor and twisted neck at 9 or10 days after IM or oral inoculation respectively.

TABLE 1 Clinical signs of birds in group 1. Clinical symptoms Helicopterchicken/ depressed/ Days post Mortality poor growth Twisted neckinoculation number/total % number/total % Number/total % 1  0/55 0  0/550  0/55 0 2  0/52 0  0/52 0  0/52 0 3  0/49 0  0/49 0  0/49 0 4  2/46 446/46 100  0/46 0 5 15/41 37 41/41 100  0/41 0 6  8/23 35 23/23 100 0/23 0 7  1/12 8 12/12 100  0/12 0 8 0/8 0 8/8 100 0/8 0 9 0/8 0 8/8100 1/8 13 10 0/8 0 8/8 100 1/8 13 11 0/6 0 6/6 100 0/6 0 12 0/6 0 6/6100 0/6 0 13 0/6 0 6/6 100 0/6 0 14 0/6 0 6/6 100 0/6 0 15 0/4 0 4/4 1000/6 0 16 0/4 0 4/4 100 0/4 0 17 0/4 0 4/4 100 0/4 0 18 0/2 0 2/2 100 0/20 19 0/2 0 2/2 100 0/2 0 20 0/2 0 2/2 100 0/2 0 21 0/2 0 2/2 100 0/2 0

TABLE 2 Clinical signs of birds in group 2.. Clinical symptomsHelicopter chicken/ depressed/ Days post Mortality poor growth Twistedneck inoculation number/total % number/total % number/total % 1 0/55 0 0/55 0 0/55 0 2 0/52 0  0/52 0 0/52 0 3 0/49 0  0/49 0 0/49 0 4 0/46 046/46 100 0/46 0 5 0/43 0 43/43 100 0/43 0 6 0/40 0 40/40 100 0/40 0 70/37 0 37/37 100 0/37 0 8 0/34 0 34/34 100 0/34 0 9 0/34 0 34/34 1000/34 0 10 1/34 3 34/34 100 1/34 3 11 0/30 0 30/30 100 0/30 0 12 0/30 030/30 100 0/30 0 13 0/30 0 30/30 100 0/30 0 14 0/30 0 30/30 100 0/30 015 0/27 0 27/27 100 0/27 0 16 0/27 0 27/27 100 0/27 0 17 0/27 0 27/27100 0/27 0 18 0/24 0 24/24 100 0/24 0 19 0/24 0 24/24 100 0/24 0 20 0/240 24/24 100 0/24 0 21 0/24 0 24/24 100 0/24 0

Serology

No antibodies were found in negative control group and no antibodiesagainst NDV, Marek's disease and AEV were found in the examinedchickens. Antibody titres against ERS were <2.0, 5.0, and 7.0 log₂ at 7,14 and 21 days of age respectively when inoculated IM and orally.

Macroscopic and Microscopic Examination

The results of the macroscopic lesions found in different organs ofchickens of group 1 and 2 are shown in Tables 1 and 2, respectively. Nomacroscopic abnormalities were found in birds of groups 3. In groups 1and 2, lesions were found in the liver (enlarged with multiple white toyellow foci), in the spleen (enlarged, hard consistence withdiscoloration), hydropericard, brains (large haemorrhage on the back ofthe cerebrum, atrophic). In group 1 lesions were also found in the legmuscle and tendon (swollen with oedema).

Microscopic examination: in the sciatic nerve a finding (apparently)related to reovirus infection was a (multi)focal inflammation withmononuclear or mixed infiltration, presence of fibroblasts, degenerationof collagen fibres, with or without small foci of necrosis. Thisinflammation was predominantly observed in adjacent (orsurrounding/interstitial/intercalating) connective tissue in few casesextending into the sciatic nerve tissue itself. Furthermore, mononuclearor heterophilic infiltration was observed and might be related toreovirus infection. In the spinal cord 3 animals with suspectedsatellitosis were found. In the connective tissue of the spinal ganglionadjacent to the spinal ganglion (or in the area of the spinal ganglionwithout evidence of it) an inflammation was observed comparable to thatin sciatic nerve. Secondly, foci of mononuclear infiltration within thespinal ganglion were observed that might also be glial cell aggregates,and also regarded as related to reovirus infection. In the sciatic nerve(central part), in only one animal an inflammation of adjacentconnective tissue as seen in the peripheral sciatic nerve. In a smallnumber of animals mononuclear infiltration in the connective tissue wasobserved. In the brain several findings were observed including 1 fociof gliosis or perivascular mononuclear infiltration, congestion ofPlexus chorioideus loops with or without presence of infiltrates.

IHC was done on the right sciatic nerve, abdominal spinal cord andbrains of birds from group 1 and on the abdominal spinal cord and brainsof birds of group 2. Positive IHC was marked as cases ofinfiltration/inflammation in connective tissue. Except in one animal ofgroup 1 at 7 dpc a ganglion cell was found positive for viral antigen.In the brains the plexus choroideus cells or related cells becamepositive from day 4 till 7 in group 1 and from day 5 till 7 in group 2.

TABLE 1 Macroscopic lesions in different organs of birds of group 1.Days post Leg muscle/ inoculation Liver Spleen hydropericard Hock jointBrains 1  0/3* 0/3 0/3 0/3 0/3 2 0/3 0/3 0/3 0/3 0/3 3 3/3 3/3 0/3 0/30/3 4 3/3 3/3 0/3 0/3 0/3 5 3/3 3/3 0/3 0/3 0/3 6 3/3 3/3 0/3 3/3 2/3 72/3 3/3 1/3 0/3 2/3 10 2/2 2/2 2/2 2/2 2/2 14 0/2 0/2 2/2 0/2 0/2 17 2/20/2 1/2 1/2 0/2 21 1/2 0/2 2/2 2/2 0/2 *Number of positive birds/totalnumber of birds

TABLE 2 Macroscopic lesions in different organs of birds of group 2.Days post Leg muscle/ Inoculation Liver Spleen Hydropericard Hock jointBrains 1  0/3* 0/3 0/3 0/3 0/3 2 0/3 0/3 0/3 0/3 0/3 3 3/3 2/3 0/3 0/30/3 4 1/3 3/3 0/3 0/3 0/3 5 1/3 1/3 0/3 0/3 0/3 6 3/3 3/3 0/3 0/3 2/3 73/3 3/3 0/3 0/3 2/3 10 3/3 2/3 1/3 0/3 0/3 14 3/3 2/3 2/3 0/3 0/3 17 1/32/3 1/3 0/3 0/3 21 0/3 0/3 0/3 0/3 0/3 *Number of positive birds/totalnumber of birds

Isolation of Reovirus

The results of Reovirus isolation from different organs of birds ofgroups 1 and 2 are demonstrated in Tables 3a and 3b respectively. Novirus was isolated from chickens of group 3. Reovirus was detected inall organs of chickens of group 1 and 2. After IM or oral inoculationisolation of virus started from 1 or 2 days after inoculationrespectively. In both groups the brains were positive till 10 days afterinoculation and the tendon stayed positive until 21 days afterinoculation.

Discussion

This report describes the pathogenesis of ERS 015 in order to reveal thepathway of ERS infecting the central nervous system. Two inoculationroutes were used, IM and orally. In both cases ERS 015 was able toinduce a twisted neck in one bird at 9 or 10 days after inoculation.These neurological symptoms can be related to the presence of virus inthe brains. ERS 015 was isolated from the brains from all examinedchickens from day 1 or 2 days after inoculation. Also viral antigen wasdemonstrated in the brains.

Histological evaluation indicates, that after IM inoculation, spread ofreovirus occurs more rapidly (lesions are seen earlier in severalorgans) and more effectively (lesions are observed during a longer timeperiod) than after oral inoculation.

Furthermore it was indicated, that nerve tissue (peripheral sciaticnerve or the surrounding connective tissue) might be a target tissue forreovirus infection with the strain ERS. Histological findings suggest,that spread of reovirus to the brain occurs via the hematogenous routerather than along nerve fibers.

To our knowledge this is the first time that an avian Reovirus isolateis capable of causing central nervous system disorders.

TABLE 3a Isolation of ERS 015 from different organs of chickens ofgroup 1. Days post Isolation of ERS 015 from inoculation Brains Legmuscle Hock joint Liver Spleen 1  0/3* 2/3 0/3 2/3 0/3 2 2/3 3/3 1/3 3/33/3 3 3/3 3/3 3/3 3/3 3/3 4 3/3 3/3 3/3 3/3 3/3 5 3/3 3/3 3/3 3/3 3/3 63/3 3/3 3/3 3/3 3/3 7 3/3 3/3 3/3 3/3 3/3 10 2/2 2/2 2/2 2/2 2/2 14 1/22/2 2/2 1/2 2/2 17 0/2 0/2 2/2 1/2 2/2 21 0/2 0/2 2/2 2/2 1/2

TABLE 3b Isolation of ERS 015 from different organs of chickens of group2. Days post Isolation of ERS 015 from inoculation Brains Leg muscleHock joint Liver Spleen 1  0/3* 0/3 0/3 0/3 0/3 2 0/3 0/3 0/3 0/3 0/3 31/3 2/3 3/3 2/3 2/3 4 1/3 2/3 2/3 2/3 2/3 5 2/3 3/3 3/3 3/3 3/3 6 3/33/3 3/3 3/3 3/3 7 3/3 3/3 3/3 3/3 3/3 10 3/3 3/3 3/3 3/3 3/3 14 0/3 1/33/3 3/3 3/3 17 0/3 2/3 3/3 3/3 3/3 21 0/2 0/2 2/2 0/2 0/2 *Number ofpositive birds/total number of birdsExperiment 6: Protection against ERS 015 Challenge of Progeny fromLayers Vaccinated with Inactivated Vaccine Containing ERS

Objective

The aim of the present study was to assess the level of protectionagainst ERS 015 challenge of progeny from layers vaccinated withinactivated vaccine containing ERS. The efficacy claim protectionagainst central nervous disorders was evaluated in this experiment.

Experimental Design

Eggs, derived from SPF-layers that were not vaccinated or vaccinatedwith inactivated ERS vaccine, were collected and hatched. Forty chicks(designated group 1) from layers vaccinated with ERS and forty chicks(designated group 2) from layers not vaccinated with ERS (180 EU/dose)were orally challenged with 0.2 ml ERS 015 (2.0 log₁₀ TCID₅₀/bird) atday old. Eight chicks (designated group 3) from layers not vaccinatedwith ERS were inoculated with 0.2 ml PBS orally and IM.

All chickens were observed daily for the occurrence of clinical signs ofdisease according to standard procedures. Special attention was takenfor central nervous disorders such as tremor and twisted neck

Five chickens of groups 1 and 2 and 1 chicken of group 3 were sacrificedat 3, 7, 10, 14, 21, 28, 35 and 42 days after challenge. Samples of thebrains were collected for virus isolation. The same procedure wasfollowed as described in experiment 5.

Interpretation of the Results

Protection against neurological symptoms was evaluated using the resultsof virus isolation for the brains. The progeny was considered protectedwhen a significant reduction in virus replication was shown in thebrains of progeny from vaccinated layers compared to progeny fromnon-vaccinated layers.

Results Clinical Symptoms

No clinical signs were seen among birds of group 3. Mortality was seenin group 1 (5 birds) and group 2 (9 birds). At necropsy enlarge liverwith multiple white spots, enlarged spleens and or hydropericard thatcould be related to reovirus infection were found in 2 of the 5 birds ofgroup 1 and in 8 of the 9 birds of group 2. Twisted neck was seen in onebird of group 1 at 14 dpc.

Isolation of Reovirus

The results of Reovirus isolation after 2 passages from the brains ofbirds are shown in Table 1. Due to the number of dead birds in groups 1and 2 it was decided to kill less birds (3 or 4) at certain necropsydates. No virus was isolated from the brains of chickens of group 1 and3. In group 2 the brains were positive till 14 days after inoculation.

TABLE 1 Isolation of ERS 015 from the brains. Isolation of ERS Days post015 from group inoculation 1 2 3 3  0/5* 5/5 0/5 7 0/4 4/4 0/5 10 0/43/3 0/5 14 0/4 0/3 0/5 21 0/4 0/3 0/5 28 0/4 0/4 0/5 35 0/5 0/4 0/5 420/5 0/5 0/5 *Number of positive birds/total number of birds

Discussion

This experiment describes the efficacy of an ERS inactivated vaccineagainst CNS disorders. It is clear that vaccination of the layers withinactivated ERS vaccine protect the progeny of an ERS infection.Mortality was lower in the birds with maternal antibodies. Only 2 birdscompare to 8 birds in the group without antibodies died from ERSinfection. Furthermore none of the birds with maternal antibodies hadvirus in the brains, while all birds of group 2 were positive till 14dpc. One chicken had a twisted neck in group I possibly a result of avaccine with too low amount of antigenic mass of ERS. No CNS was seen ingroup 2 but on the other hand, 9 of the 40 birds died within 13 dpc andthis is the time that twisted necks can be developed. It could be thatthose chickens past away before nervous symptoms could appear. It can beconcluded based on mortality and ERS isolation from the brains thatanti-ERS maternal antibodies protect the progeny from CNS disorderscaused by ERS.

For a further understanding of the scope of the present invention,consideration should be had to the appended Claims.

1. A method of treating and/or preventing neurological symptoms inpoultry caused by an avian reovirus comprises administering an effectiveamount of an immunogenic composition or vaccine to the poultrycomprising an avian reovirus that causes neurological symptoms and apharmaceutically acceptable carrier or diluent.
 2. The method of claim 1wherein the reovirus is in a live, attenuated or killed form.
 3. Themethod of claim 1 wherein the avian reovirus is an Enteric ReovirusStrain (ERS) belonging to a class of reovirus that is able to induceantiserum in an animal, which antiserum causes a reduction of theplaques formed by strain ERS, a sample of which is deposited at theECACC, Salisbury, UK, under accession no. 99011475, of at least 75% in aplaque reduction assay.
 4. The method of claim 3 wherein the reductionof plaques formed by strain ERS is at least 80% in a plaque reductionassay.
 5. The method of claim 3 wherein the reduction of the plaquesformed by strain ERS is at least 90% in a plaque reduction assay.
 6. Themethod of claim 1 or 3 wherein the reovirus is characterized byreactivity in an immuno-fluorescence-technique (IFT) with a polyclonalantiserum raised against a second avian reovirus isolate; and, theabsence of reactivity in an IFT with Moabs INT 13-06, INT 14-11, and15-01 INT (samples of which are deposited at the ECACC under accessionnos. 99011472, 99011473, and 9901474).
 7. The method of claim 6 whereinthe second avian reovirus is selected from the group consisting ofstrain S1133; strain 2408; strain 1733; strain 2177; strain ERS; strainERS 060E; strain ERS 074; and strain ERS
 1037. 8. The method of claim 1wherein the immunogenic composition or vaccine further comprises avaccine strain of at least one of Marek's Disease Virus, InfectiousBursal Disease Virus, Newcastle Disease Virus, Infectious BronchitisVirus, Avian Encephalomyelitis Virus, Fowl Pox Virus, and Chicken AnemiaAgent.
 9. The method of claim 1 wherein the immunogenic composition orvaccine further comprises an adjuvant.
 10. The method of claim 1 whereinthe neurological symptoms are selected from the group consisting oftremors and twisted neck.
 11. The method of claim 1 wherein the avianreovirus is selected from the group consisting of strain ERS (isolate1), strain ERS 060E, strain ERS 074, and strain ERS
 1037. 12. The methodof claim 1 wherein the poultry is selected from the group consisting ofchickens, turkeys, water fowl, guineas, quail, pigeons, and ostriches.13. An immunogenic composition or vaccine for preventing neurologicalsymptoms comprising an enteric reovirus strain (ERS) and a carrier ordiluent.
 14. The immunogenic composition or vaccine of claim 13 whereinthe ERS is selected from the group consisting of strain ERS (isolate 1),strain ERS 060E, strain ERS 074, and strain ERS
 1037. 15. Theimmunogenic composition or vaccine of claim 13 wherein the immunogeniccomposition or vaccine further comprises a vaccine strain of at leastone of Marek's Disease Virus, Infectious Bursal Disease Virus, NewcastleDisease Virus, Infectious Bronchitis Virus, Avian EncephalomyelitisVirus, Fowl Pox Virus, and Chicken Anemia Agent.
 16. A method ofisolating an enteric reovirus strain from poultry comprising the stepsof a. Obtaining at least a portion of the neurological system of thepoultry; and, b. Isolating an avian reovirus from the neurologicalsystem.
 17. A class of avian reovirus that causes neurological symptomsin poultry.
 18. The class of claim 17 wherein the reovirus is an entericreovirus strain (ERS).
 19. The class of claim 17 wherein the ERS isselected from the group consisting of strain ERS (isolate 1), strain ERS060E, strain ERS 074, and strain ERS 1037.